A kind of tubulin depolymerization agent polypeptide and its application
A tubulin and depolymerizing agent technology, applied to tubulin depolymerizing agent polypeptide 1 and its application field, can solve the problems of lack of specificity of small molecule depolymerizing agents, and achieve the effects of increasing the survival rate and improving the survival rate.
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Embodiment 1
[0017] Effect of tubulin depolymerizer polypeptide 1 on tubulin polymerization and depolymerization in vitro.
[0018] Take fresh bovine brain tissue, peel off the meninges and large blood vessels, cut into pieces, wash 1-2 times with cold MES buffer, add MES buffer at a ratio of 0.5-1ml per gram of brain tissue, and at 4°C, use electric Homogenize with a homogenizer; centrifuge at 105,000 g for 1 h at 4°C, take the supernatant, add an equal volume of microtube polymerization buffer, and incubate in a water bath at 37°C for 30 min. Centrifuge at 105,000 g at 26°C for 1 hour, take the precipitate, add about 1 / 10 of the homogenate volume of cold MES buffer, stir gently or crush the precipitate with a homogenizer; place the suspension in an ice bath for 30 min to dissolve the precipitate completely. Protein content was determined by Lowry's method (SDS polyamide gel electrophoresis). Tubulin was diluted to 4-5 mg / ml with MES buffer and stored in liquid nitrogen. Take the frozen...
Embodiment 2
[0022] IC50 of tubulin depolymerizer polypeptide 1 on the growth and survival of tumor cells cultured in vitro.
[0023] The MTT colorimetric method was used. The logarithmic growth of U937 cells was added to a 96-well culture plate at 1.0×105, and cultured for 24 hours. Different concentrations of the experimental drug tubulin depolymerizer polypeptide 1 and the positive control drug Changchun Xin were added to the experimental wells and positive drug control wells, respectively. base; add the same volume of solvent to the blank group. Set up five duplicate wells per well, culture for 48h, add MTT to each well at 0h, 2h, 8h, 14h, 20h, 24h, 36h, and 48h respectively, add DMSO after 4h of action, incubate for 30min, and incubate at 620nm in a microplate reader Measure the absorbance A value, according to the formula tumor cell growth inhibition rate=(1-absorbance value of the experimental group / absorbance value of the control group)×100%. The IC50 of the experimental drug was...
Embodiment 3
[0025] In vivo activity of tubulin depolymerizer polypeptide 1 was tested in a tumor model.
[0026] The U937 tumor model was established, and the positive control drug was vincristine; the same volume of solvent was added to the blank group, and 3 doses were set to the experimental group: 0.75, 1.5 μM, and 3 μM mg / Kg. After 21 days, the number of surviving mice was observed, and the survival rate was calculated. The results showed that the tubulin depolymerizing agent polypeptide 1 could effectively protect mice and improve the survival rate of tumor-bearing mice, with the survival rate reaching 73.2%.
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